role of STAT2 in type I interferon-induced responses.
Published: 2004
Language: eng
Number of pages: 257
Description: STAT2 plays a key role in the Type I Interferon (IFN) signaling cascade. Upon IFN-alpha stimulation, two distinct types of STAT2-containing complexes are formed: IFN-stimulated gene factor 3 (ISGF3), and ISGF3-independent STAT2-containing complexes. In the ISGF3 complex, STAT1 and IRF9 directly mediate IFN-stimulated response element (ISRE) binding, while STAT2 provides a potent transactivational domain. Conversely, while ISGF3-independent STAT2-containing complexes, specifically STAT2:STAT1 and STAT2:STAT3, bind a gamma-activated sequence- (GAS)-like pIRE, the contribution of each STAT protein to DNA binding is unknown. To examine the role of STAT2 in mediating IFN-inducible responses in target cells, we generated a series of STAT2 mutant cDNAs. These cDNAs were introduced by transfection into U6A cells lacking STAT2, resulting in a panel of cell lines that expressed the various mutant STAT2 proteins. Studies were undertaken to assess the sensitivity of U6A cells reconstituted with intact STAT2 (U6A-STAT2) and cells expressing mutant STAT2s (U6A-STAT2E427,428A, U6A-STAT2V453I, U6A-STAT2V454I, U6A-STAT2V454A, U6A-STAT2V453,4541, U6A-STAT2N458A) to IFN-inducible responses. Data reveal that, in response to IFN-a treatment, cell types expressing both intact and mutant STAT2 exhibit normal ISGF3 and STAT2:STAT1 complex formation and DNA binding. However, luciferase gene reporter assays performed to assess the transcriptional activation potential of these STAT2-containing complexes revealed that cells expressing the V453,4541 mutant of STAT2 are impaired in their ability to drive GAS-mediated transcription. Moreover, whereas cells expressing STAT2 mutants E427,428A, V453I, V4541, V454A and N458A are responsive to the antiviral and growth inhibitory effects of IFN-alpha but U6A-STAT2V453,454I cells are refractory to these IFN-inducible biological activities. Finally, gene expression microarray analysis and tandem mass spectroscopy protein expression profiling identified specific targets whose expression is dependent upon IFN-induction of a fully functional STAT2 protein thereby highlighting the importance of ISGF3-independent STAT2-containing complexes in IFN-induced responses.
Notes: Adviser: Eleanor Fish.

Thesis (Ph.D.)--University of Toronto, 2004.

Electronic version licensed for access by U. of T. users.

Source: Dissertation Abstracts International, Volume: 65-10, Section: B, page: 5056.